Hla-a* 3303-restricted wt1 peptide and pharmaceutical composition comprising the same

ABSTRACT

Disclosed are: a peptide comprising an amino acid sequence composed of contiguous nine amino acid residues derived from a WT1 protein, wherein an amino acid residue at position 2 in the amino acid sequence is selected from the group consisting of Ala, Ile, Leu, Val, Phe, Tyr, Ser and Asp and an amino acid residue at position 9 in the amino acid sequence is Arg; a polynucleotide encoding the peptide; a pharmaceutical composition comprising the peptide; and others.

TECHNICAL FIELD

The present invention relates to an HLA-A*3303-restricted peptide,specifically a peptide comprising an amino acid sequence consisting of 9contiguous amino acids from a WT1 protein, wherein an amino acid atposition 2 of the amino acid sequence is selected from the groupconsisting of Ala, Ile, Leu, Val, Phe, Tyr, Ser and Asp, and an aminoacid at position 9 is Arg. Furthermore, the present invention relates toa polynucleotide encoding the peptide, a pharmaceutical composition forthe treatment or prevention of a cancer comprising the same, and thelike.

BACKGROUND

WT1 gene (Wilms' tumor 1 gene) was identified as a gene responsible forWilms tumor which is a renal cancer in children (Non-patent Documents 1and 2). WT1 is a transcription factor having a zinc finger structure. Atthe beginning, the WT1 gene was considered to be a tumor suppressorgene. However, subsequent studies (Non-patent Documents 3, 4, 5 and 6)showed that the WT1 gene rather functions as an oncogene inhematopoietic tumors and solid cancers.

The WT1 gene is expressed at high levels in many types of malignanttumors. It has been examined whether or not the WT1 gene product free ofmutations, which is an autologous protein, has immunogenicity in aliving body. The results revealed that the protein derived from the WT1gene which is expressed at high levels in tumor cells is fragmentedthrough intracellular processing, the resulting peptides form complexeswith MHC class I molecules, and the complexes are presented on thesurfaces of cells, and that CTLs recognizing such complexes can beinduced by peptide vaccination (Non-patent Documents 7, 8 and 9). It wasalso shown that in a mouse immunized with a WT1 peptide or a WT1 cDNA,transplanted tumor cells expressing a WT1 gene are rejected with a highprobability (Non-patent Documents 7 and 10), while normal tissuesexpressing physiologically the WT1 gene are not damaged by the inducedCTLs (Non-patent Document 7). It was shown in in vitro experiments usinghuman cells that when Db126 peptide or WH187 peptide (amino acids187-195 of SEQ ID No: 1, SLGEQQYSV) having a high ability to bind to anHLA-A^(*)0201 molecule, which is one of human MHC class I molecules, isused to stimulate human peripheral blood mononuclear cells havingHLA-A*0201, WT1-specific CTLs are induced, the induced CTLs have acytotoxic activity specific for tumor cells expressing endogenously aWT1 gene at high levels, and the cytotoxic activity of such CTLs isHLA-A2-restricted (Non-patent Document 11). It was shown in in vitroexperiments in human cells using WT1 peptide that matches HLA-A*2402(which is found most frequently in Japanese people among HLA-A alleles)(WT1₂₃₅; amino acids 235-243 of SEQ ID No: 1, CMTWNQMNL) thatWT1-specific CTLs (TAK-1) are induced (Non-patent Document 12), and theinduced CTLs do not suppress the colony-forming activity of normalhematopoietic stem cells which partially express physiologically a WT1gene (Non-patent Documents 13 and 14). These reports strongly suggestthat not only in mice but also in human beings, WT1-specific CTLs can beinduced, such CTLs have a cytotoxic activity against tumor cellsexpressing a WT1 gene at high levels, but do not have a cytotoxicactivity against normal cells expressing physiologically a WT1 gene(Non-patent Documents 7, 10, 11, 12, 13 and 14).

The WT1 gene product is present as a nuclear protein, and is processedby proteasomes in cytoplasm to be fragmented into peptides. Thefragmented peptides are transported into endoplasmic reticulum lumen byTAP (transporter associated with antigen processing) molecules, formcomplexes with MHC class I molecules, and are presented on the surfacesof cells. WT1-specific CTLs are induced as a result of recognition ofWT1 peptide-MHC class I molecule complexes by CTL precursor cells viaTCR, thereby exerting a cytotoxic effect on tumor cells presenting a WT1gene product through MHC class I molecules (Non-patent Documents 7, 8and 9). Then, it is required at least that a WT1 peptide used in cancerimmunotherapy targeting a WT1 gene product is in the form that binds toan MHC class I molecule in a living body. However, MHC class I moleculesare diverse and amino acid sequences of the WT1 peptides binding torespective MHC class I molecules are different from each other.Therefore, it is required to provide a peptide matching each subtype ofMHC class I. However, only HLA-A*2402 molecule-, HLA-A*0201 molecule-and HLA-A*2601 molecule-restricted peptides are known as HLAmolecule-restricted WT1 peptides to date (Patent Document 1, Non-patentDocument 11 and Patent

Document 2, respectively). HLA-A*3303 is present at next highestpercentage to HLA-A*2402 in Japanese. Therefore, there is a need to findan HLA-A*3303-restricted WT1 peptide.

Patent Document 1: WO 2003/106682

Patent Document 2: WO 2005/095598

Non-patent Document 1: Daniel A. Haber et al., Cell. 1990 Jun. 29;61(7):1257-69.

Non-patent Document 2: Call K M et al., Cell. 1990 Feb. 9; 60(3):509-20.

Non-patent Document 3: Menke A L et al., Int Rev Cytol. 1998;181:151-212. Review.

Non-patent Document 4: Yamagami T et al., Blood. 1996 Apr. 1;87(7):2878-84.

Non-patent Document 5: Inoue K et al., Blood. 1998 Apr. 15;91(8):2969-76.

Non-patent Document 6: Tsuboi A et al., Leuk Res. 1999 May;23(5):499-505.

Non-patent Document 7: Oka Y et al., J Immunol. 2000 Feb. 15;164(4):1873-80.

Non-patent Document 8: Melief C J et al., Immunol Rev. 1995 June;145:167-77.

Non-patent Document 9: Ritz J, J Clin Oncol. 1994 February; 12(2):237-8.

Non-patent Document 10: Tsuboi A et al., J Clin Immunol. 2000 May;20(3):195-202.

Non-patent Document 11: Oka Y et al., Immunogenetics. 2000 February;51(2):99-107.

Non-patent Document 12: Ohminami H et al., Blood. 2000 Jan. 1;95(1):286-93.

Non-patent Document 13: Gao L et al., Blood. 2000 Apr. 1;95(7):2198-203.

Non-patent Document 14: Ohminami H et al., Blood. 2000 Jan. 1;95(1):286-93.

DISCLOSURE OF INVENTION Problems to be Solved by the Invention

The problems to be solved by the present invention are to provide anHLA-A*3303-restricted WT1 peptide, and a polynucleotide encoding thesame, as well as a pharmaceutical composition for thetreatment/prevention of a cancer comprising the same, and the like.

Means to Solve the Problems

As a result of intensive studies in view of the situation as describedabove, the present inventor has found that a peptide comprising an aminoacid sequence consisting of 9 contiguous amino acids from a WT1 protein,wherein an amino acid at position 2 of the amino acid sequence isselected from the group consisting of Ala, Ile, Leu, Val, Phe, Tyr, Serand Asp, and an amino acid at position 9 of the amino acid sequence isArg can induce a WT1-specific CTL with a high rate. Thus, the presentinvention has been completed.

The present invention provide:

(1) a peptide comprising an amino acid sequence consisting of 9contiguous amino acids from a WT1 protein, wherein an amino acid atposition 2 of the amino acid sequence is selected from the groupconsisting of Ala, Ile, Leu, Val, Phe, Tyr, Ser and Asp, and an aminoacid at position 9 of the amino acid sequence is Arg:

(2) the peptide according to (1), wherein the amino acid sequence isselected from the group consisting of:

Leu Ser His Leu Gln Met His Ser Arg, (SEQ ID No: 2)Phe Ser Arg Ser Asp Gln Leu Lys Arg, (SEQ ID No: 3)Ser Asp Gln Leu Lys Arg His Gln Arg, (SEQ ID No: 4) andThr Ser Glu Lys Pro Phe Ser Cys Arg; (SEQ ID No: 5)

(3) the peptide according to (2), wherein the amino acid sequence is SerAsp Gln Leu Lys Arg His Gln Arg (SEQ ID No: 4);

(4) a pharmaceutical composition for the treatment or prevention of acancer, comprising the peptide according to (1);

(5) a method for the treatment or prevention of a cancer, comprisingadministering an effective amount of the pharmaceutical compositionaccording to (4) to an HLA-A*3303-positive subject;

(6) use of the peptide according to (1) for the manufacture of thepharmaceutical composition according to (4);

(7) a polynucleotide encoding the peptide according to (1);

(8) an expression vector comprising the polynucleotide according to (7);

(9) a pharmaceutical composition for the treatment or prevention of acancer, comprising the polynucleotide according to (7) or the vectoraccording to (8) ;

(10) a method for the treatment or prevention of a cancer, comprisingadministering an effective amount of the pharmaceutical compositionaccording to (9) to an HLA-A*3303-positive subject;

(11) use of the polynucleotide according to (7) or the vector accordingto (8) for the manufacture of the pharmaceutical composition accordingto (9);

(12) a WT1-specific CTL, which is inducible by the peptide according to(1);

(13) a method for the induction of a WT1-specific CTL, comprisingculturing a peripheral blood mononuclear cell in the presence of thepeptide according to (1) to induce the WT1-specific CTL from theperipheral blood mononuclear cell;

(14) a kit for the induction of a WT1-specific CTL, comprising thepeptide according to (1) as an essential component;

(15) an antigen-presenting cell presenting a WT1 peptide, which isinducible by the peptide according to (1);

(16) a method for the induction of an antigen-presenting cell presentinga WT1 peptide, comprising culturing an immature antigen-presenting cellin the presence of the peptide according to (1) to induce theantigen-presenting cell presenting a WT1 peptide from the immatureantigen-presenting cell;

(17) a kit for the induction of an antigen-presenting cell presenting aWT1 peptide, comprising the peptide according to (1) as an essentialcomponent;

(18) a method for the diagnosis of a cancer, comprising using the CTLaccording to (12) or the antigen-presenting cell according to (15);

(19) a method for the determination of the presence or amount of aWT1-specific CTL in an HLA-A*3303-positive subject, comprising:

-   (a) reacting a complex of a WT1 peptide and an HLA-A*3303 molecule    with a sample from the subject; and-   (b) determining the presence or amount of a CTL recognizing the    complex contained in the sample; and

(20) the method according to (19), wherein the complex is a form oftetramer.

Effects of the Invention

The present invention provides an HLA-A*3303-restricted WT1 peptide, anda polynucleotide encoding the same, as well as a pharmaceuticalcomposition for the treatment or prevention of a cancer comprising thesame, and the like. Therefore, it is possible to induce in vivo and invitro WT1-specific CTLs in subjects having HLA-A*3303. Because about 24%of Japanese people have at least one HLA-A*3303 molecule, WT1-specificCTLs can be induced in a very wide range of subjects.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 represents the cytotoxic activity of the CTL induced with WT1₃₃₇.

FIG. 2 represents the cytotoxic activity of the CTL induced with WT1₃₆₄.

FIG. 3 represents the cytotoxic activity of the CTL induced with WT1₃₆₇.

FIG. 4 represents the cytotoxic activity of the CTL induced with WT1₄₀₉.

FIG. 5 represents the cytotoxic activity of the CTL induced with WT1₃₆₄against the cell expressing a WT1 gene endogenously.

FIG. 6 represents the cytotoxic activity of the CTL induced with WT1₃₆₇against the cell expressing a WT1 gene endogenously.

FIG. 7 represents the cytotoxic activity of the CTL induced with WT1₄₀₉against the cell expressing a WT1 gene endogenously.

FIG. 8 represents the cytotoxic activity of the CTL induced with WT1₃₆₇against the cell expressing a WT1 gene.

BEST MODE FOR CARRYING OUT THE INVENTION

An amino acid sequence of a human WT1 protein is shown in SEQ ID No: 1.A WT1 gene is expressed in its native form at high levels, for example,in hematopoietic tumors such as leukemia, myelodysplastic syndrome,multiple myeloma or malignant lymphoma and solid cancers such as gastriccancer, colon cancer, lung cancer, breast cancer, germ cell cancer,hepatic cancer, skin cancer, bladder cancer, prostate cancer, uterinecancer, cervical cancer or ovarian cancer. Furthermore, an anchor motiffor HLA-A*3303 is characterized in that an amino acid at position 2 isany one of Ala, Ile, Leu, Val, Phe, Tyr, Ser and Asp, and an amino acidat position 9 is Arg. Thus, in one aspect, the present invention relatesto an HLA-A*3303-restricted WT1 peptide comprising an amino acidsequence consisting of 9 contiguous amino acids from a WT1 protein,wherein an amino acid at position 2 of the amino acid sequence ispreferably selected from the group consisting of Ala, Ile, Leu, Val,Phe, Tyr, Ser and Asp, and an amino acid at position 9 of the amino acidsequence is preferably Arg (hereinafter referred to as a WT1 peptide).

The amino acid sequence consisting of 9 amino acids comprised in thepeptide of the present invention is preferably, Leu Ser His Leu Gln MetHis Ser Arg (SEQ ID No: 2), Phe Ser Arg Ser Asp Gln Leu Lys Arg (SEQ IDNo: 3), Ser Asp Gln Leu Lys Arg His Gln Arg (SEQ ID No: 4) or Thr SerGlu Lys Pro Phe Ser Cys Arg (SEQ ID No: 5). Most preferably, it is SerAsp Gln Leu Lys Arg His Gln Arg (SEQ ID No: 4). Furthermore, it may havea substitution of one to several, preferably one to five amino acidswith other amino acids in the 9 amino acids of any of SEQ ID Nos: 2-5.Any one of the 9 amino acids or other substituted amino acids may beappropriately modified. In any cases, the peptide of the presentinvention retains an ability to bind to an HLA-A*3303 molecule.

As described above, it is an object of the present invention to obtainan HLA-A*3303-restricted WT1 peptide. Thus, the peptide of the presentinvention may be any one as long as it comprises an amino acid sequencethat is derived from a WT1 protein and consists of 9 contiguous aminoacids. Thus, the peptide of the present invention may be, for example, apeptide consisting of only the amino acid sequence shown in any of SEQID Nos: 2-5, or a WT1 protein or a part thereof comprising the aminoacid sequence shown in any of SEQ ID Nos: 2-5. Various substances may beattached at the N-terminus and/or the C-terminus of the amino acidsequence consisting of 9 contiguous amino acids in the peptide of thepresent invention. For example, an amino acid, a peptide or an analogthereof may be attached. If these substances are attached to the peptideof the present invention, they can be processed, for example, by anenzyme in a living body or through a process such as intracellularprocessing, and finally the amino acid sequence consisting of 9contiguous amino acids can be produced and presented as a complex withan HLA-A*3303 molecule on the surface of a cell, thereby resulting inthe effect of inducing a CTL. These substances may be those whichmodulate the solubility of the peptides of the present invention, orincrease their stability (resistance to protease, etc.). Alternatively,these substances may be those which deliver the peptides of the presentinvention specifically, for example, to a given tissue or organ, or theymay have the action to increase the efficiency of uptake by anantigen-presenting cell or the like. These substances may be those whichincrease the ability to induce a CTL, such as helper peptides or thelike.

The peptide of the present invention can be synthesized by methodsgenerally used in the art or modifications thereof. Such methods aredescribed, for example, in Peptide Synthesis, Interscience, New York,1966; The Proteins, Vol 2, Academic Press Inc., New York, 1976;Peptide-Gosei, Maruzen Co., Ltd., 1975; Peptide-Gosei No Kiso To Jikken,Maruzen Co., Ltd., 1985; and Iyakuhin No Kaihatsu (Zoku), Vol. 14,Peptide-Gosei, Hirokawa—Book store, 1991.

The peptide of the present invention can also be prepared using geneticengineering techniques based on the information about the nucleotidesequence that encodes the peptide of the present invention. Such geneticengineering techniques are well known to a person skilled in the art.

In another aspect, the present invention relates to a pharmaceuticalcomposition for the treatment or prevention of a cancer, comprising theHLA-A*3303-restricted WT1 peptide. The WT1 gene is expressed at highlevels in hematopoietic tumors such as leukemia, myelodysplasticsyndrome, multiple myeloma or malignant lymphoma, and solid cancers suchas gastric cancer, colon cancer, lung cancer, breast cancer, germ cellcancer, hepatic cancer, skin cancer, bladder cancer, prostate cancer,uterine cancer, cervical cancer or ovarian cancer. Therefore, thepharmaceutical composition of the present invention can be used for thetreatment or prevention of a cancer. When the pharmaceutical compositionof the present invention is administered to an HLA-A*3303-positivesubject, WT1-specific CTLs are induced by the HLA-A*3303-restricted WT1peptide comprised in the pharmaceutical composition, and cancer cells inthe subject are damaged by such CTLs.

The pharmaceutical composition of the present invention may comprise inaddition to the HLA-A*3303-restricted WT1 peptide as an activeingredient, for example, a carrier, an excipient or the like. TheHLA-A*3303-restricted WT1 peptide comprised in the pharmaceuticalcomposition of the present invention induces a WT1-specific CTL. Thus,the pharmaceutical composition of the present invention may comprise anappropriate adjuvant, or may be administered together with anappropriate adjuvant in order to enhance the induction efficiency.Examples of preferable adjuvants include, but are not limited to,complete or incomplete Freund's adjuvant and aluminum hydroxide.

The method of the administration of the pharmaceutical composition ofthe present invention can be appropriately selected depending onconditions such as the type of disease, the condition of the subject orthe target site. Examples of such methods include, but are not limitedto, intradermal administration, subcutaneous administration,intramuscular administration, intravenous administration, nasaladministration and oral administration. The amount of the peptidecomprised in the pharmaceutical composition of the present invention, aswell as the dosage form, the number of times of the administration andthe like of the pharmaceutical composition of the present invention canbe appropriately selected depending on conditions such as the type ofdisease, the condition of the subject or the target site. The singledose of the peptide is usually, 0.0001 mg-1000 mg, preferably, 0.001mg-10000 mg.

In another aspect, the present invention relates to a method for thetreatment or prevention of a cancer, comprising administering aneffective amount of the pharmaceutical composition to anHLA-A*3303-positive subject. The cancer to be treated or prevented maybe any one, and examples thereof include hematopoietic tumors such asleukemia, myelodysplastic syndrome, multiple myeloma or malignantlymphoma and solid cancers such as gastric cancer, colon cancer, lungcancer, breast cancer, germ cell cancer, hepatic cancer, skin cancer,bladder cancer, prostate cancer, uterine cancer, cervical cancer orovarian cancer.

In another aspect, the present invention relates to use of anHLA-A*3303-restricted WT1 peptide for the manufacture of thepharmaceutical composition.

In a further aspect, the present invention relates to a method for thedetermination of the presence or amount of a WT1-specific CTL in anHLA-A*3303-positive subject, comprising:

-   (a) reacting a complex of a WT1 peptide and an HLA-A*3303 molecule    with a sample form the subject; and-   (b) determining the presence or amount of a CTL recognizing the    complex contained in the sample. The sample from a subject may be    any one as long as there is a possibility that it contains a    lymphocyte. Examples of the samples include body fluid such as blood    or lymph and a tissue. The complex of a WT1 peptide and an    HLA-A*3303 molecule may be prepared, for example, as a tetramer or    pentamer using a method known to a person skilled in the art such as    biotin-streptavidin method. The presence or amount of the CTL    recognizing such a complex can be measured by a method known to a    person skilled in the art. In this aspect of the present invention,    the complex may be labeled. A known label such as a fluorescent    label or a radioactive label can be used as a label. Labeling makes    the determination of the presence or amount of a CTL easy and rapid.    The method of this aspect of the present invention can be used to    diagnose a caner, prognosis thereof or the like.

Thus, the present invention also provides a composition for thedetermination of the presence or amount of a WT1-specific CTL in anHLA-A*3303-positive subject, comprising a complex of a WT1 peptide andan HLA-A*3303 molecule.

Furthermore, the present invention provides a kit for the determinationof the presence or amount of a WT1-specific CTL in anHLA-A*3303-positive subject, comprising a complex of a WT1 peptide andan HLA-A*3303 molecule.

In a further aspect, the present invention relates to a method for theproduction of a WT1-specific CTL using a complex of a WT1 peptide and anHLA-A*3303 molecule, comprising:

-   (a) reacting the complex with a sample; and-   (b) obtaining a CTL recognizing the complex contained in the sample.    The complex of a WT1 peptide and an HLA-A*3303 molecule is described    above. The sample may be any one as long as there is a possibility    that it contains a lymphocyte. Examples of the samples include a    sample from a subject such as blood, and a cell culture. The CTL    recognizing the complex can be obtained using a method known to a    person skilled in the art such as FACS or MACS. The present    invention allows to culture the obtained WT1-specific CTL and use it    for the treatment or prevention of various cancers.

Thus, the present invention also relates to a

WT1-specific CTL which is obtainable by a method for the production of aWT1-specific CTL using a complex of a WT1 peptide and an HLA-A*3303molecule.

Furthermore, the present invention relates to a kit for the productionof a WT1-specific CTL, comprising a complex of a WT1 peptide and anHLA-A*3303 molecule.

In another aspect, the present invention relates to a polynucleotideencoding the HLA-A*3303-restricted WT1 peptide (hereinafter referred toas a WT1 polynucleotide). The polynucleotide of the present inventionmay be DNA or RNA. The base sequence of the polynucleotide of thepresent invention can be determined based on the amino acid sequence ofthe HLA-A*3303-restricted WT1 peptide. The polynucleotide can beprepared, for example, by a method for the synthesis of DNA or RNA, PCRmethod or the like.

In another aspect, the present invention relates to an expression vectorcomprising the polynucleotide (hereinafter referred to as a WT1expression vector). The type of the expression vector, the comprisedsequence other than the sequence of the polynucleotide and the like canbe appropriately selected depending on the type of a host into which theexpression vector of the present invention is introduced, the purpose ofuse, or the like. It is possible to treat or prevent hematopoietictumors or solid cancers by administering the expression vector of thepresent invention to an HLA-A*3303-positive subject to produce a WT1peptide in a living body and induce a WT1-specific CTL, and damaginghematopoietic tumor cells or solid cancer cells in the subject.

In another aspect, the present invention relates to a pharmaceuticalcomposition for the treatment or prevention of a cancer, comprising theWT1 polynucleotide or the WT1 expression vector. The composition, themethod of the administration and the like of the pharmaceuticalcomposition of the present invention in this aspect are described above.

In another aspect, the present invention relates to a method for thetreatment or prevention of a cancer, comprising administering aneffective amount of the pharmaceutical composition comprising the WT1peptide or the WT1 expression vector to an HLA-A*3303-positive subject.Examples of cancers to be treated or prevented include hematopoietictumors such as leukemia, myelodysplastic syndrome, multiple myeloma ormalignant lymphoma and solid cancers such as gastric cancer, coloncancer, lung cancer, breast cancer, germ cell cancer, hepatic cancer,skin cancer, bladder cancer, prostate cancer, uterine cancer, cervicalcancer or ovarian cancer.

In another aspect, the present invention relates to use of a WT1polynucleotide or a WT1 expression vector for the manufacture of thepharmaceutical composition comprising the WT1 polynucleotide or the WT1expression vector.

In another aspect, the present invention relates to a cell comprisingthe expression vector. The cell of the present invention can beprepared, for example, by transforming a host cell such as E. coli,yeast, insect cell or animal cell with the expression vector. The methodfor the introduction of the expression vector into a host cell can beappropriately selected from various methods. By culturing thetransformed cell, and recovering and purifying the produced WT1 peptide,the peptide of the present invention can be prepared.

In a further aspect, the present invention relates to a WT1-specificCTL, which is induced by the HLA-A*3303-restricted WT1 peptide. The CTLof the present invention recognizes a complex of a WT1 peptide and anHLA-A*3303 molecule. Thus, the CTL of the present invention can be usedto damage specifically a tumor cell positive for HLA-A*3303 andexpressing WT1 at a high level.

In another aspect, the present invention relates to a method for thetreatment or prevention of a cancer, comprising administering aWT1-specific CTL to an HLA-A*3303-positive subject. The method of theadministration of the WT1-specific CTL can be appropriately selecteddepending on conditions such as the type of the disease, the conditionof the subject or the target site. Examples of such methods include, butare not limited to, intravenous administration, intradermaladministration, subcutaneous administration, intramuscularadministration, nasal administration and oral administration.

In another aspect, the present invention relates to a method for theinduction of a WT1-specific CTL, comprising culturing a peripheral bloodmononuclear cell in the presence of the HLA-A*3303-restricted WT1peptide to induce the WT1-specific CTL form the peripheral bloodmononuclear cell. The subject from which the peripheral bloodmononuclear cell is derived may be any one as long as it is positive forHLA-A*3303. By culturing the peripheral blood mononuclear cells in thepresence of the HLA-A*3303-restricted WT1 peptide, WT1-specific CTLs areinduced from CTL precursor cells contained in the peripheral bloodmononuclear cells. It is possible to treat or prevent hematopoietictumors or solid cancers in an HLA-A*3303-positive subject byadministering the WT1-specific CTL obtained according to the presentinvention to the subject.

In another aspect, the present invention relates to a kit for theinduction of a WT1-specific CTL, comprising an HLA-A*3303-restricted WT1peptide as an essential component. Preferably, the kit is used in themethod for the induction of a WT1-specific CTL. The kit of the presentinvention may comprise in addition to the HLA-A*3303-restricted WT1peptide, for example, a means of obtaining a peripheral bloodmononuclear cell, an adjuvant, a reaction vessel or the like. Ingeneral, an instruction manual is attached to the kit. By using the kitof the present invention, WT1-specific CTLs can be induced efficiently.

In a further aspect, the present invention relates to anantigen-presenting cell (such as a dendritic cell) presenting a WT1peptide through an HLA-A*3303 molecule, which is induced by theHLA-A*3303-restricted WT1 peptide. By using the antigen-presenting cellof the present invention, WT1-specific CTLs are induced efficiently.

In another aspect, the present invention relates to a method for thetreatment or prevention of a cancer, comprising administering theantigen-presenting cell presenting a WT1 peptide through an HLA-A*3303molecule to an HLA-A*3303-positive subject. The method of theadministration of the antigen-presenting cell can be appropriatelyselected depending on conditions such as the type of the disease, thecondition of the subject or the target site. Examples of such methodsinclude, but are not limited to, intravenous administration, intradermaladministration, subcutaneous administration, intramuscularadministration, nasal administration and oral administration.

In another aspect, the present invention relates to a method for theinduction of an antigen-presenting cell presenting a WT1 peptide throughan HLA-A*3303 molecule, comprising culturing an immatureantigen-presenting cell in the presence of the HLA-A*3303-restricted WT1peptide to induce the antigen-presenting cell presenting a WT1 peptidethrough an HLA-A*3303 molecule from the immature antigen-presentingcell. The immature antigen-presenting cell refers a cell such as animmature dendritic cell that can be matured into an antigen-presentingcell. A subject from which the immature antigen-presenting cell isderived may be any one as long as it is positive for HLA-A*3303. Becausethe immature antigen-presenting cells are contained, for example, inperipheral blood mononuclear cells, such cells may be cultured in thepresence of the WT1 peptide.

In another aspect, the present invention relates to a kit for theinduction of an antigen-presenting cell presenting a WT1 peptide throughan HLA-A*3303 molecule, comprising the HLA-A*3303-restricted WT1 peptideas an essential component. Preferably, the kit is used in the method forthe induction of an antigen-presenting cell. Another component to becomprised in the kit of the present invention and the like are describedabove. The kit of the present invention can be used to induceefficiently an antigen-presenting cell presenting a WT1 peptide throughan HLA-A*3303 molecule.

In another aspect, the present invention relates to an antibody againstan HLA-A*3303-restricted WT1 peptide or an antibody against apolynucleotide encoding the peptide. The antibody of the presentinvention may be a polyclonal antibody or monoclonal antibody.

In a further aspect, the present invention relates to a method for thediagnosis of a cancer, comprising using the WT1-specific CTL, theantigen-presenting cell presenting a WT1 peptide through an HLA-A*3303molecule, or the antibody against an HLA-A*3303-restricted WT1 peptideor the antibody against a polynucleotide encoding the peptide.Preferably, the WT1-specific CTL is used in the method for the diagnosisof the present invention. For example, it is possible to diagnose acancer by incubating the CTL, the antigen-presenting cell or theantibody with a sample from an HLA-A*3303-positive subject, oradministering it to an HLA-A*3303-positive subject, and determining, forexample, the position, site or amount thereof. The CTL, theantigen-presenting cell or the antibody may be labeled. By attaching alabel, the method for the diagnosis of the present invention can bepracticed efficiently.

In another aspect, the present invention relates to a kit for thediagnosis of a cancer, comprising the WT1-specific CTL, theantigen-presenting cell presenting a WT1 peptide through an HLA-A*3303molecule, or the antibody against an HLA-A*3303-restricted WT1 peptideor the antibody against a polynucleotide encoding the peptide as anessential component.

The following examples illustrate the present invention in more detail,but are not to be construed to limit the scope thereof.

Examples Example 1

Selection of WT1 Peptide

NetMHC2.0 Program (Technical University of Denmark) was used to selectWT1₃₃₇, WT1₃₆₄, WT1₃₆₇ and WT1₄₀₉, which are hydrophilic peptidesconsisting of 9 amino acids with the anchor motif suitable forHLA-A*3303 (the second amino acid from the N-terminus is any one of Ala,Ile, Leu, Val, Phe, Tyr, Ser and Asp, and the amino acid at theC-terminus is Arg) and are expected to have a high binding affinity toan HLA-A*3303 molecule from a WT1 peptide from a WT1 protein (SEQ ID No:1). Amino acid sequences and the binding affinities to an HLA-A*3303molecule of these peptides are shown in Table 1.

TABLE 1 Binding Amino acid  afinity to Number in Amino acid HLA-A*3303Peptide SEQ ID NO: 1 sequence molecule WT1₃₃₇ 337-345 LSHLQMHSR 18.827(SEQ ID No: 2) WT1₃₆₄ 364-372 FSRSDQLKR 15.143 (SEQ ID No: 3) WT1₃₆₇367-375 SDQLKRHQR 14.496 (SEQ ID No: 4) WT1₄₀₉ 409-417 TSEKPFSCR 15.310(SEQ ID No: 5)

Preparation of B-LCL Cell

Peripheral blood mononuclear cells (PBMCs) were separated byFicoll-Hypaque gradient density centrifugation method from peripheralblood that had been collected from an HLA-A*3303-positive healthy donor(HLA-A*3303/0207). The PBMCs were then seeded to a 24-well cell cultureplate at the density of about 1×10⁷ in RPMI 1640 medium containing 10%FCS, and a culture supernatant of B95-8 cells (cells producing EB virus)were added. They were cultured at 37° C. with 5% CO₂ for about 1 month.B-LCL cells transformed with EB virus, which are B-cell tumor cells,were obtained. It was confirmed that the resulting B-LCL cells did notexpress WT1 gene. B-LCL cells were pulsed by incubating them with 20μg/ml of WT1₃₃₇, WT1₃₆₄, WT1₃₆₇ or WT1₄₀₉ for 2 hours, and irradiatedwith 80 Gy of radiation. The resulting B-LCL cells (hereinafter referredto as B-LCL cells pulsed with a WT1 peptide) were used asantigen-presenting cells for the following experiments.

Induction of CTL Specific WT1

3×10⁶ of PBMCs (HLA-A*3303/1101) were cultured in a 24-well cell cultureplate in complete medium (45% RPMI, 45% AMI-V medium and 10% human ABserum) containing 20 μg/ml of WT1₃₃₇, WT1₃₆₄, WT1₃₆₇ or WT1₄₀₉ at 37° C.with 5% CO₂ for 1 week to obtain responding cells. 2×10⁶ of theresulting responding cells were cocultured with 1×10⁶ of the B-LCL cellspulsed with the same WT1 peptide in complete medium for 1 week (firststimulation). The PBMCs were cocultured with the B-LCL cells pulsed withthe WT1 peptide three more times (second to fourth stimulations) underthe conditions under which 20 IU/ml (final concentration) of IL-2 wasadded as follows: second stimulation: two times every other day from 3days after the initiation of stimulation; third and fourth stimulations:three times at intervals of one day from the day after the initiation ofstimulation. The resulting cells were concentrated using NegativeSelection Columns Gravity Feed Kit (StemSp) so that the ratio ofCD8-positive T cells became about 80%, and cocultured with the B-LCLcells pulsed with the WT1 peptide (fifth stimulation). CD8-positive Tcells (CTLs) obtained 5 days after the final stimulation were used formeasurement of the cytotoxic activity.

Cytotoxic Activity of CTL

The cytotoxic activity of CTLs was measured using ⁵¹Cr release assay.CTL cells (hereinafter referred to as effector cells) were mixed at theratio (E/T ration) of 1:1, 5:1 or 10:1 in 200 μl of medium with targetcells into which ⁵¹Cr had been incorporated, and cultured in a 96-wellcell culture plate at 37° C. with 5% CO₂ for 4 hours. B-LCL cells pulsedwith the same WT1 peptide as that used for induction of CTLs (BLCL-Ps),and B-LCL cells without pulsing with a WT1 peptide (BLCL-NPs) were usedas target cells. After the culture, the supernatants were collected bycentrifugation. The amounts of ⁵¹Cr released into the supernatants weremeasured using a liquid scintillation counter. The cytotoxic activity(%) was determined using the following formula:

(⁵¹Cr release in supernatant of sample−Spontaneous ⁵¹Crrelease)/(Maximum ⁵¹Cr release−Spontaneous ⁵¹Cr release)×100

wherein Spontaneous ⁵¹Cr release is ⁵¹Cr release observed when thetarget cells into which ⁵¹Cr had been incorporated were cultured aloneunder the same condition, and Maximum ⁵¹Cr release is ⁵¹Cr releaseobserved when the target cells into which ⁵¹Cr had been incorporatedwere completely lysed using 1% Triton X-100. Results are shown in FIGS.1-4. In the figures, longitudinal axes represent specific lysis (%), andhorizontal axes represent E/T ratios. BLCL-Ps are represented using fulllines, and BLCL-NPs are represented using dotted lines. It was confirmedthat CTLs induced with WT1₃₃₇, WT1₃₆₄, WT1₃₆₇ or WT1₄₀₉ damagespecifically BLCL-Ps presenting the WT1 peptide as a complex with anHLA-A*3303 molecule as compared with BLCL-NPs. CTLs induced with WT1₃₆₄,WT1₃₆₇ or WT1₄₀₉ were used for further experiments below.

Cytotoxic Activity of CTL Against Cell Expressing WT1 Gene Endogenously

The cytotoxic activity of CTLs induced with WT1₃₆₄, WT1₃₆₇ or WT1₄₀₉against TF-1 cells that are tumor cells expressing a WT1 gene(HLA-A*3303-positive) was determined using the method as describedabove. As a control, K562 cells that express a WT1 gene and are negativefor HLA-A*3303 were used. Results are shown in FIGS. 5-7. In thefigures, longitudinal axes represent specific lysis (%), and horizontalaxes represent E/T ratios. TF-1 is represented using full lines, andK562 is represented using dotted lines. It was confirmed that CTLsinduced with WT1₃₆₄, WT1₃₆₇ or WT1₄₀₉ also have a cytotoxic activityagainst the cell expressing a WT1 gene exogenously.

The cytotoxic activity of CTLs induced with WT1₃₆₇ against B-LCL cellexpressing WT1 was determined using the method as described above. TheB-LCL expressing WT1 (B-LCL-WT1) refers to B-LCL cell into which a humanWT1 gene is introduced and expresses a WT1 protein in the cell, andpresents a peptide consisting of about 9 amino acids resulting fromprocessing on an HLA-A*3303 molecule. As a control, B-LCL cell(B-LCL-CV) into which a control gene except a WT1 gene is introduced wasused. Results are shown in FIG. 8. In the figures, longitudinal axesrepresent specific lysis (%), and horizontal axes represent E/T ratios.B-LCL-WT1 is represented using full lines, and B-LCL-CV is representedusing dotted lines. It was confirmed that CTLs induced with WT1₃₆₇damage only the cell which is HLA-A*3303-positive and expresses WT1.

INDUSTRIAL APPLICABILITY

The present invention provides an HLA-A*3303-restricted WT1 peptide, apolynucleotide encoding the peptide, a pharmaceutical compositioncomprising the same and the like. Therefore, the present invention canbe used in the fields of medicine and the like, for example, in thefields of development and preparation of a pharmaceutical compositionfor the prevention or treatment of various hematopoietic tumors andsolid cancers that express WT1 gene at high levels.

1. A peptide comprising an amino acid sequence consisting of 9contiguous amino acids from a WT1 protein, wherein an amino acid atposition 2 of the amino acid sequence is selected from the groupconsisting of Ala, Ile, Leu, Val, Phe, Tyr, Ser and Asp, and an aminoacid at position 9 of the amino acid sequence is Arg.
 2. The peptideaccording to claim 1, wherein the amino acid sequence is selected fromthe group consisting of: Leu Ser His Leu Gln Met His Ser Arg,(SEQ ID No: 2) Phe Ser Arg Ser Asp Gln Leu Lys Arg, (SEQ ID No: 3)Ser Asp Gln Leu Lys Arg His Gln Arg, (SEQ ID No: 4) andThr Ser Glu Lys Pro Phe Ser Cys Arg. (SEQ ID No: 5)


3. The peptide according to claim 2, wherein the amino acid sequence isSer Asp Gln Leu Lys Arg His Gln Arg (SEQ ID No: 4).
 4. A pharmaceuticalcomposition for the treatment or prevention of a cancer, comprising thepeptide according to claim
 1. 5. A method for the treatment orprevention of a cancer, comprising administering an effective amount ofthe pharmaceutical composition according to claim 4 to anHLA-A*3303-positive subject.
 6. Use of the peptide according to claim 1for the manufacture of the pharmaceutical composition according to claim4.
 7. A polynucleotide encoding the peptide according to claim
 1. 8. Anexpression vector comprising the polynucleotide according to claim
 7. 9.A pharmaceutical composition for the treatment or prevention of acancer, comprising the polynucleotide according to claim 7 or the vectoraccording to claim
 8. 10. A method for the treatment or prevention of acancer, comprising administering an effective amount of thepharmaceutical composition according to claim 9 to anHLA-A*3303-positive subject.
 11. Use of the polynucleotide according toclaim 7 or the vector according to claim 8 for the manufacture of thepharmaceutical composition according to claim
 9. 12. A WT1-specific CTL,which is inducible by the peptide according to claim
 1. 13. A method forthe induction of a WT1-specific CTL, comprising culturing a peripheralblood mononuclear cell in the presence of the peptide according to claim1 to induce the WT1-specific CTL from the peripheral blood mononuclearcell.
 14. A kit for the induction of a WT1-specific CTL, comprising thepeptide according to claim 1 as an essential component.
 15. Anantigen-presenting cell presenting a WT1 peptide, which is inducible bythe peptide according to claim
 1. 16. A method for the induction of anantigen-presenting cell presenting a WT1 peptide, comprising culturingan immature antigen-presenting cell in the presence of the peptideaccording to claim 1 to induce the antigen-presenting cell presenting aWT1 peptide from the immature antigen-presenting cell.
 17. A kit for theinduction of an antigen-presenting cell presenting a WT1 peptide,comprising the peptide according to claim 1 as an essential component.18. A method for the diagnosis of a cancer, comprising using the CTLaccording to claim 12 or the antigen-presenting cell according to claim15.
 19. A method for the determination of the presence or amount of aWT1-specific CTL in an HLA-A*3303-positive subject, comprising: (a)reacting a complex of a WT1 peptide and an HLA-A*3303 molecule with asample from the subject; and (b) determining the presence or amount of aCTL recognizing the complex contained in the sample.
 20. The methodaccording to claim 19, wherein the complex is a form of tetramer.